CN110507635A - It is a kind of using hyaluronic acid decorated graphene oxide as the preparation method of the Adriamycin nanoparticles of carrier - Google Patents

It is a kind of using hyaluronic acid decorated graphene oxide as the preparation method of the Adriamycin nanoparticles of carrier Download PDF

Info

Publication number
CN110507635A
CN110507635A CN201910929625.9A CN201910929625A CN110507635A CN 110507635 A CN110507635 A CN 110507635A CN 201910929625 A CN201910929625 A CN 201910929625A CN 110507635 A CN110507635 A CN 110507635A
Authority
CN
China
Prior art keywords
added
solution
water
reaction
graphene oxide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201910929625.9A
Other languages
Chinese (zh)
Inventor
王秉
徐城锋
胡锦华
黄芊蔚
胡智文
万军民
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University of Technology ZJUT
Zhejiang Sci Tech University ZSTU
Zhejiang University of Science and Technology ZUST
Original Assignee
Zhejiang University of Technology ZJUT
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University of Technology ZJUT filed Critical Zhejiang University of Technology ZJUT
Priority to CN201910929625.9A priority Critical patent/CN110507635A/en
Publication of CN110507635A publication Critical patent/CN110507635A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The present invention relates to field of medical materials, it discloses a kind of using hyaluronic acid decorated graphene oxide as the preparation method of the Adriamycin nanoparticles of carrier, graphene oxide is prepared first using crystalline flake graphite as raw material, by improving Hummers method and a series of post-processings in the present invention.Using ethylenediamine as linking arm, hyaluronic acid is converted to HA-NH using the amidation process between carboxyl and amino2, then activated with coupling agent, HA-NH2It modifies on graphene oxide, to obtain HA-GO.Adriamycin is added in backward HA-GO solution and obtains Adriamycin nanoparticles after reaction and post-processing.The nanoparticle of this method preparation will not make a significant impact cell in the experimentation in later period, not influence the science of experimental result, and preparation flow is simple and convenient to operate, does not need harsh reaction condition and special reaction unit.

Description

It is a kind of using hyaluronic acid decorated graphene oxide as the Adriamycin nanoparticles of carrier Preparation method
Technical field
The present invention relates to field of medical materials more particularly to a kind of using hyaluronic acid decorated graphene oxide as the Ah of carrier The preparation method of mycin nanoparticle.
Background technique
New derivatives of the graphene oxide as graphene, by people's extensive concern.It is a kind of tightly packed at six The carbon allotrope of the two-dimensional crystal lattice of triangular arrangement is by complete graphite area and some sp3 hydbridized carbon atoms such as carboxylics The carbon atom composition of sp2 hydridization in base, hydroxyl and epoxy group etc. and phenyl ring.Because of its intrinsic unique texture and physics Mechanical and optical property, GO show great potentiality, such as cell imaging in biological medicine and biological applications, and photo-thermal is controlled Treatment and bio-pharmaceutical device.
GO makes it in biotechnology, biomedical engineering, nanosecond medical science, tumour with unique machinery, electronics, optical property Treatment, organizational project, drug release, bio-imaging and biomolecule sensing etc. have all played huge effect.But GO's Bio-safety problem makes it cause certain limitation in clinical application, cytotoxicity, toxicity in vivo including them, heredity Toxicity and the bioconcentration in certain organs (such as lung and liver) all need further to be studied.With the hair of material science Exhibition, we will, biocompatibility better material low with toxicity modify GO, to prepare, property is stable, structure is bright Really, safe and non-toxic GO enters it among more extensive clinical research as safely and effectively medical material.
Summary of the invention
In order to solve the above-mentioned technical problems, the present invention provides a kind of using hyaluronic acid decorated graphene oxide as carrier The preparation method of Adriamycin nanoparticles, the present invention is first using crystalline flake graphite as raw material, by improving hummers method and a series of Graphene oxide is prepared in post-processing.Using ethylenediamine as linking arm, the amidation process between carboxyl and amino is utilized Hyaluronic acid is converted to HA-NH2, then activated with coupling agent, HA-NH2It modifies on graphene oxide, to obtain HA- GO.Adriamycin is added in backward HA-GO solution and obtains Adriamycin nanoparticles after reaction and post-processing.This method preparation Nanoparticle will not make a significant impact cell in the experimentation in later period, not influence the science of experimental result, and make Standby process is simple and convenient to operate, does not need harsh reaction condition and special reaction unit.
The specific technical proposal of the invention is: a kind of using hyaluronic acid decorated graphene oxide as the adriamycin nano of carrier The preparation method of particle, comprising the following steps:
1) container is cooled to 0-5 DEG C in ice-water bath, the 40-50mL concentrated sulfuric acid is added；Then 1-4g crystalline flake graphite is added Powder and 0.4-2g sodium nitrate；Ultrasonic treatment.
2) it is stirred to react, keeps reaction temperature to be lower than 10 DEG C, then weigh 4-8g potassium permanganate, in 25-30min in batches 1.5-2.5h is reacted in secondary addition.
The present invention increases potassium manganate in batches can make reaction more mild, convenient for control temperature of reaction system, prevent anti- Should be excessively fierce, generate unnecessary impurity.Simultaneously graphite-sulfuric acid single order can be generated under its oxidation by sulfuric acid intercalation Intercalation object.
3) it is warming up to 35-40 DEG C, reacts 25-30min.
4) 140-240mL deionized water is measured, is added after ice-water bath is cooled to 0-5 DEG C；Then temperature adjustment is to 90-95 DEG C, continue to be stirred to react 25-30min.
The present invention is by graphite oxide obtained in step 3), by being reacted with water, while water enter interlayer substitution it is therein Acid obtains graphene oxide to removing.
5) 100-140mL water stopped reaction is added, adds the H that 40-60mL volumetric concentration is 25-35%2O2Solution, after It is continuous to be stirred to react 15-20min.
6) HCl solution that 60-100mL volumetric concentration is 8-12% is added, stands 4-5 days.
Product is stood 4-5 days by the present invention, penetrates into the separation of the impurity such as the acid in lower sediment in supernatant, to subtract Few centrifugation time.
7) supernatant of solution, Zai Qu lower layer viscous fluid centrifugal treating are first removed, until viscous fluid is in neutrality, by viscous fluid Freeze-drying, obtains graphene oxide sheet.
8) graphene oxide sheet is ground into graphene oxide powder, i.e. GO powder.
9) claim HA 150-200mg in container, dissolved with 10-15mL formamide, to container closure, 45-55 DEG C of oil bath is extremely Dissolution.
10) HA solution is cooled to room temperature, weighs EDC 500-540mg respectively, NHS 300-320mg uses 8- respectively It is added in HA solution after the formamide vortex dissolution of 12mL, 3-7mL, activation-COOH 25-35min is stirred at room temperature, is activated HA solution.
11) it will be placed in container dissolved with the 10-15mL formamide solution of 1-1.5mL ethylenediamine, use constant pressure under condition of ice bath Dropping funel instills the HA solution of activation in gained ethylenediamine solution, and 50-70min is added dropwise under condition of ice bath.
The present invention has obtained HA-NH by the amidation process of ethylenediamine and hyaluronic acid2。
12) reaction system is warmed to room temperature reaction 2-4h.
13) acetone that the quality of pre-cooling is 3-5 times of reaction solution is added under ice bath after reaction, cooling, crystallization is stood, Precipitating, 0.22 μm of organic film filter, and acetone washing precipitating is removed precipitating, redissolved with water, dialysed in water, every 6-8h replacement one Secondary water is freeze-dried up to intermediate product HA-NH after the 48-60h that dialyses2。
The present invention is filtered using organic film, is dialysed using the bag filter of MWCO=10000-14000Da, the centre made Product purity is higher.
14) it weighs GO powder 45-60mg to be placed in container, 30-40mL formamide is added, ice bath shifts after visiting super 25-35min Into another container, and previous container is rinsed with 20-30mL formamide and flushing liquor is transferred in latter container.
15) EDC 170-175mg, NHS 100-105mg are weighed, and is dissolved respectively with 5-10mL formamide, then by it It is added in the latter container of step 14), activation 10-20min, the GO solution activated is stirred at room temperature.
16) 90-135mg HA-NH is weighed2It is dissolved in 10-15mL formamide, and 180-270 μ L triethylamine is added, wait live It is added dropwise to after change in the GO solution of activation, reacts at room temperature 20-30h.
Step 14), step 15) in step 16), are activated with carboxyl of the EDC and NHS to GO, are improved amidated Coupling efficiency.
17) quality that pre-cooling is added under ice bath after reaction is 3-5 times of acetone of reaction solution, and cooling, crystallization is quiet It sets, precipitates, 0.22 μm of organic film filters, and acetone washing precipitating is removed precipitating, redissolved with water, dialysed in water, every 4-6h replacement Water, dialysis 48-60h obtain HA-GO solution.
18) adriamycin that 0.8-1mL concentration is 0.8-1.2mg/mL is taken to instill in HA-GO solution；It is protected from light stirring 24-30h, Centrifugation collects particle, saves after the material freeze of lower layer is dry, target product is made.
In step 18), is combined by the carboxyl of HA-GO with adriamycin, Adriamycin nanoparticles have been made.
Preferably, being ultrasonically treated 20-30min in step 1).
Preferably, in step 2), stirring rate 400-800rpm stirs 20-40min.
Preferably, during standing, every 1-2d removes supernatant in step 6), aliquots of deionized water is added.
Preferably, being freeze-dried 24-48h in step 7).
Preferably, in step 7), centrifugal process specifically: respectively in 3500-4500rpm, 5500-6500rpm, It is respectively centrifuged 5-15min under 7500-8500rpm, 9000-11000rpm, 11000-13000rpm, removes supernatant after centrifugation every time Simultaneously aliquots of deionized water is added in liquid.
Preferably, in step 13) and step 17), the bag filter for the use of specification being MWCO=10000-14000Da.
It is compared with the prior art, the beneficial effects of the present invention are: the present invention passes through improvement first using crystalline flake graphite as raw material Graphene oxide is prepared in hummers method and a series of post-processings.Using ethylenediamine as linking arm, carboxyl and ammonia are utilized Hyaluronic acid is converted to HA-NH by the amidation process between base2, then activated with coupling agent, HA-NH2Modify graphite oxide On alkene, to obtain HA-GO.Adriamycin is added in backward HA-GO solution and obtains adriamycin nano after reaction and post-processing Particle.The nanoparticle of this method preparation will not make a significant impact cell in the experimentation in later period, not influence to test As a result science, and preparation flow is simple and convenient to operate, does not need harsh reaction condition and special reaction unit.
Detailed description of the invention
Fig. 1 is the XRD spectra of obtained graphene oxide during embodiment 1.
Fig. 2 is that hyaluronic acid decorated graphene oxide of the invention carries out transmission electron microscope (TEM) phenogram.
Specific embodiment
The present invention will be further described with reference to the examples below.
Embodiment 1
1) beaker is cooled to 0 DEG C in ice-water bath, magneton is added, is added to 250mL with the concentrated sulfuric acid that graduated cylinder measures 40mL In beaker；Then it weighs 1g crystalline graphite powder and beaker is added in 0.4g sodium nitrate；Then ultrasound is carried out, 20min is continued；
2) beaker in step 1) is placed in magnetic agitation water-bath, 20min is stirred under the revolving speed of 400rpm, keep anti- It answers temperature lower than 10 DEG C, then weighs 4g potassium permanganate on an electronic balance, be slowly added in batches in 25min；The stage React 1.5h；
3) water bath temperature in step 2) is risen to 35 DEG C, reacts 25min；
4) 140mL deionized water is measured slowly to be added in step 3) in beaker after ice-water bath is cooled to 0 DEG C；Then Regulating step 3) water bath temperature is to after 90 DEG C or so, the reaction was continued under stirring auxiliary 25min；
5) into step 4), the distilled water stopped reaction of 100mL is added in beaker, and adding 40mL volume fraction is 30% H2O2Solution continues to be stirred to react 15min；
6) HCl solution that 60mL volume fraction is 10% is measured with graduated cylinder, is then added into beaker in step 5).It will produce Object stands 4d in beaker, and every 1d removes supernatant, and aliquots of deionized water is added；
7) supernatant in step 6) in beaker first being removed with dropper, Zai Qu lower layer viscous fluid uses supercentrifuge, point It is not centrifuged 10min at 4000rpm, 6000rpm, 8000rpm, 10000rpm, 12000rpm, removes centrifuge tube after centrifugation every time Simultaneously aliquots of deionized water is added in middle supernatant, until graphene oxide viscous fluid is in neutrality, the viscous fluid in centrifuge tube is poured into steaming It sends out in ware, freeze-drying obtains graphene oxide sheet for 24 hours；
8) by graphene oxide sheet grind into powder in step 7)；
9) claim HA 150mg in 50mL flask, dissolved with 10mL formamide, glass stopper sealing, 50 DEG C of oil baths to dissolution, Obtain HA solution；
10) HA solution is cooled to room temperature, is ratio with-COOH: EDC: NHS=1: 5: 5, weigh EDC 520mg, NHS 310mg uses 10mL respectively, is added in HA solution after the formamide vortex dissolution of 5mL, activation-COOH 30min is stirred at room temperature；
11) it will be placed in 100mL flask dissolved with the 10mL formamide solution of 1mL ethylenediamine, dripped under condition of ice bath with constant pressure The HA of activation is slowly dropped into above-mentioned ethylenediamine solution by liquid funnel, and 60min is added dropwise under condition of ice bath；
12) reaction system is warmed to room temperature reaction 3h；
13) excessive propanone (4 times of reaction solution) of pre-cooling is added under ice bath after reaction, cooling, crystallization is stood, and is sunk Forming sediment is HA-NH2, 0.22 μm of organic film filters, and acetone washing precipitating is removed precipitating, redissolved with water, uses bag filter (MWCO =12000Da) it dialyses in water, every 6h replaces a water, is freeze-dried after the 48h that dialyses up to intermediate product HA-NH2；
14) it weighs GO powder 45mg to be placed in 50mL beaker, 30mL formamide is added, ice bath is transferred to after visiting super 30min In 150mL round-bottomed flask, and beaker is rinsed with 20mL formamide and is transferred in flask；
15) EDC 173mg, NHS 103mg are weighed, and is dissolved respectively with 5mL formamide, above-mentioned flask is then added into In, activation 15min is stirred at room temperature；
16) 90mg HA-NH is weighed2Be dissolved in 10mL formamide, and be added 180 μ L triethylamines, it is to be activated after by its Fast drop is into the GO solution of activation, and room temperature reaction is for 24 hours；
17) excessive propanone (4 times of reaction solution) of pre-cooling is added under ice bath after reaction, cooling, crystallization is stood, and is sunk Forming sediment is HA-GO, and 0.22 μm of organic film filters, and acetone washing precipitating is removed precipitating, redissolved with water, uses bag filter (MWCO= It 12000Da) dialyses in water, every 4h replaces a water, and dialysis 48h obtains HA-GO solution；
18) taking 0.8mL concentration is that the adriamycin of 2mg/mL is slowly dropped into HA-GO solution；It is protected from light stirring 30h, is centrifuged, is received Collect particle, is saved after the material freeze of lower layer is dry, Adriamycin nanoparticles are made.
Fig. 1 is the XRD spectrum of graphene oxide, and natural graphite has characteristic peak at 26 ° of 2 θ ≈, after peroxidating, oxygen Peak of the graphite alkene at 26 ° disappears, and mobile to low angle, the strong diffraction maximum at one newly occurs at 11 ° of 2 θ ≈ places, and 11 ° 001 and 100 crystal faces are corresponded respectively to 42 °.It is approximately according to the interplanar distance that bragg's formula can calculate this oxidation product 0.98nm.For raw graphite, its interplanar distance is significantly increased after oxidation, shows the generation of graphite oxide.
Transmission electron microscope (TEM) characterization is carried out to hyaluronic acid decorated graphene oxide, TEM result as shown in Fig. 2, by Hyaluronic acid decorated graphene oxide still keeps lamellar structure, i.e. modificationization effect occurs over just surface of graphene oxide On active group, do not change the structure of graphene itself.This also illustrates hyaluronic acids successfully to modify in graphene oxide Surface can play the role of a targeting.
Infrared detection is carried out to product, graphite powder is oxidized to after graphene oxide, and a large amount of oxygen-containing groups occurs in surface, Such as epoxy group, carbonyl, carboxyl.It is characterized according to the measurement of infrared spectroscopy: 1720cm-1The stretching vibration for carbonyl occur absorbs Peak, 1621cm-1There is the stretching vibration absworption peak of C-O-C, in 1388cm-1The stretching vibration for epoxy group occur absorbs Peak, after functional modification hyaluronic acid, in 2923cm-1、1307cm-1、1161cm-1Occurs the spy of hyaluronic acid respectively Levy absorption peak.
Embodiment 2
1) beaker is cooled to 0 DEG C in ice-water bath, magneton is added, is added to 250mL with the concentrated sulfuric acid that graduated cylinder measures 45mL In beaker；Then it weighs 2g crystalline graphite powder and beaker is added in 1g sodium nitrate；Then ultrasound is carried out, 5min is continued；
2) beaker in step 1) is placed in magnetic agitation water-bath, 30min is stirred under the revolving speed of 600rpm, keep anti- It answers temperature lower than 10 DEG C, then weighs 6g potassium permanganate on an electronic balance, be slowly added in batches in 28min；The stage React 2h；
3) water bath temperature in step 2) is risen to 38 DEG C, reacts 28min；
4) 190mL deionized water is measured slowly to be added in step 3) in beaker after ice-water bath is cooled to 0 DEG C；Then Regulating step 3) water bath temperature is to after 93 DEG C or so, the reaction was continued under stirring auxiliary 27min；
5) into step 4), the distilled water stopped reaction of 120mL is added in beaker, and adding 50mL volume fraction is 30% H2O2Solution continues to be stirred to react 18min；
6) HCl solution that 80mL volume fraction is 10% is measured with graduated cylinder, is then added into beaker in step 5).It will produce Object stands 4.5d in beaker, and every 1.5d removes supernatant, and aliquots of deionized water is added；
7) supernatant in step 6) in beaker first being removed with dropper, Zai Qu lower layer viscous fluid uses supercentrifuge, point It is not centrifuged 10min at 4000rpm, 6000rpm, 8000rpm, 10000rpm, 12000rpm, removes centrifuge tube after centrifugation every time Simultaneously aliquots of deionized water is added in middle supernatant, until graphene oxide viscous fluid is in neutrality, the viscous fluid in centrifuge tube is poured into steaming It sends out in ware, freeze-drying 36h obtains graphene oxide sheet；
8) by graphene oxide sheet grind into powder in step 7)；
9) claim HA 175mg in 50mL flask, dissolved with 13mL formamide, glass stopper sealing, 50 DEG C of oil baths to dissolution, Obtain HA solution；
10) HA solution is cooled to room temperature, is ratio with-COOH: EDC: NHS=1: 5: 5, weighs EDC520mg, NHS310mg uses 12mL respectively, is added in HA solution after the formamide vortex dissolution of 6mL, activation-COOH is stirred at room temperature 30min；
11) it will be placed in 100mL flask dissolved with the 13mL formamide solution of 1.3mL ethylenediamine, use constant pressure under condition of ice bath The HA of activation is slowly dropped into above-mentioned ethylenediamine solution by dropping funel, and 60min is added dropwise under condition of ice bath；
12) reaction system is warmed to room temperature reaction 3h；
13) excessive propanone (4 times of reaction solution) of pre-cooling is added under ice bath after reaction, cooling, crystallization is stood, and is sunk Forming sediment is HA-NH2, 0.22 μm of organic film filters, and acetone washing precipitating is removed precipitating, redissolved with water, uses bag filter (MWCO =12000Da) it dialyses in water, every 7h replaces a water, is freeze-dried after the 56h that dialyses up to intermediate product HA-NH2；
14) it weighs GO powder 53mg to be placed in 50mL beaker, 35mL formamide is added, ice bath is transferred to after visiting super 30min In 150mL round-bottomed flask, and beaker is rinsed with 25mL formamide and is transferred in flask；
15) EDC 173mg, NHS 103mg are weighed, and is dissolved respectively with 8mL formamide, above-mentioned flask is then added into In, activation 15min is stirred at room temperature；
16) 113mg HA-NH is weighed2It is dissolved in 13mL formamide, and 225 μ L triethylamines is added, it will after to be activated Its fast drop is into the GO solution of activation, and room temperature reaction is for 24 hours；
17) excessive propanone (4 times of reaction solution) of pre-cooling is added under ice bath after reaction, cooling, crystallization is stood, and is sunk Forming sediment is HA-GO, and 0.22 μm of organic film filters, and acetone washing precipitating is removed precipitating, redissolved with water, uses bag filter (MWCO= It 12000Da) dialyses in water, every 5h replaces a water, and dialysis 50h obtains HA-GO solution；
18) taking 0.9mL concentration is that the adriamycin of 2mg/mL is slowly dropped into HA-GO solution；It is protected from light stirring 27h, is centrifuged, is received Collect particle, is saved after the material freeze of lower layer is dry, Adriamycin nanoparticles are made.
Embodiment 3
1) beaker is cooled to 0 DEG C in ice-water bath, magneton is added, is added to 250mL with the concentrated sulfuric acid that graduated cylinder measures 50mL In beaker；Then it weighs 4g crystalline graphite powder and beaker is added in 2g sodium nitrate；Then ultrasound is carried out, 30min is continued；
2) beaker in step 1) is placed in magnetic agitation water-bath, 40min is stirred under the revolving speed of 800rpm, keep anti- It answers temperature lower than 10 DEG C, then weighs 8g potassium permanganate on an electronic balance, be slowly added in batches in 30min；The stage React 2.5h；
3) water bath temperature in step 2) is risen to 40 DEG C, reacts 30min；
4) 240mL deionized water is measured slowly to be added in step 3) in beaker after ice-water bath is cooled to 0 DEG C；Then Regulating step 3) water bath temperature is to after 95 DEG C or so, the reaction was continued under stirring auxiliary 30min；
5) into step 4), the distilled water stopped reaction of 140mL is added in beaker, and adding 60mL volume fraction is 30% H2O2Solution continues to be stirred to react 20min；
6) the HCI solution that 100mL volume fraction is 10% is measured with graduated cylinder, is then added into beaker in step 5).It will Product stands 5d in beaker, and every 2d removes supernatant, and aliquots of deionized water is added；
7) supernatant in step 6) in beaker first being removed with dropper, Zai Qu lower layer viscous fluid uses supercentrifuge, point It is not centrifuged 10min at 4000rpm, 6000rpm, 8000rpm, 10000rpm, 12000rpm, removes centrifuge tube after centrifugation every time Simultaneously aliquots of deionized water is added in middle supernatant, until graphene oxide viscous fluid is in neutrality, the viscous fluid in centrifuge tube is poured into steaming It sends out in ware, freeze-drying 48h obtains graphene oxide sheet；
8) by graphene oxide sheet grind into powder in step 7)；
9) claim HA 200mg in 50mL flask, dissolved with 15mL formamide, glass stopper sealing, 50 DEG C of oil baths to dissolution, Obtain HA solution；
10) HA solution is cooled to room temperature, is ratio with-COOH: EDC: NHS=1: 5: 5, weigh EDC 520mg, NHS 310mg uses 10mL respectively, is added in HA solution after the formamide vortex dissolution of 5mL, activation-COOH30min is stirred at room temperature；
11) it will be placed in 100mL flask dissolved with the 15mL formamide solution of 1.5mL ethylenediamine, use constant pressure under condition of ice bath The HA of activation is slowly dropped into above-mentioned ethylenediamine solution by dropping funel, and 60min is added dropwise under condition of ice bath；
12) reaction system is warmed to room temperature reaction 3h；
13) excessive propanone (4 times of reaction solution) of pre-cooling is added under ice bath after reaction, cooling, crystallization is stood, and is sunk Forming sediment is HA-NH2, 0.22 μm of organic film filters, and acetone washing precipitating is removed precipitating, redissolved with water, uses bag filter (MWCO =12000Da) it dialyses in water, every 8h replaces a water, is freeze-dried after the 60h that dialyses up to intermediate product HA-NH2；
14) it weighs GO powder 60mg to be placed in 50mL beaker, 40mL formamide is added, ice bath is transferred to after visiting super 30min In 150mL round-bottomed flask, and beaker is rinsed with 30mL formamide and is transferred in flask；
15) EDC 173mg, NHS 103mg are weighed, and is dissolved respectively with 10mL formamide, above-mentioned burning is then added into In bottle, activation 15min is stirred at room temperature；
16) 135mg HA-NH is weighed2It is dissolved in 15mL formamide, and 270 μ L triethylamines is added, it will after to be activated Its fast drop is into the GO solution of activation, and room temperature reaction is for 24 hours；
17) excessive propanone (4 times of reaction solution) of pre-cooling is added under ice bath after reaction, cooling, crystallization is stood, and is sunk Forming sediment is HA-GO, and 0.22 μm of organic film filters, and acetone washing precipitating is removed precipitating, redissolved with water, uses bag filter (MWCO= It 12000Da) dialyses in water, every 6h replaces a water, and dialysis 60h obtains HA-GO solution；
18) taking 1mL concentration is that the adriamycin of 2mg/mL is slowly dropped into HA-GO solution；It is protected from light stirring 30h, is centrifuged, is collected Particle saves after the material freeze of lower layer is dry, Adriamycin nanoparticles is made.
Raw materials used in the present invention, equipment is unless otherwise noted the common raw material, equipment of this field；In the present invention Method therefor is unless otherwise noted the conventional method of this field.
The above is only presently preferred embodiments of the present invention, is not intended to limit the invention in any way, it is all according to the present invention Technical spirit any simple modification, change and equivalent transformation to the above embodiments, still fall within the technology of the present invention side The protection scope of case.

Claims (7)

1. it is a kind of using hyaluronic acid decorated graphene oxide as the preparation method of the Adriamycin nanoparticles of carrier, with mg, g and mL Meter, it is characterised in that the following steps are included:
1) container is cooled to 0-5 DEG C in ice-water bath, the 40-50mL concentrated sulfuric acid is added；Then be added 1-4g crystalline graphite powder with 0.4-2g sodium nitrate；Ultrasonic treatment；
2) it is stirred to react, keeps reaction temperature to be lower than 10 DEG C, then weigh 4-8g potassium permanganate, add in batches in 25-30min Enter, reacts 1.5-2.5h；
3) it is warming up to 35-40 DEG C, reacts 25-30min；
4) 140-240mL deionized water is measured, is added after ice-water bath is cooled to 0-5 DEG C；Then temperature adjustment is to 90-95 DEG C, after It is continuous to be stirred to react 25-30min；
5) 100-140mL water stopped reaction is added, adds the H that 40-60mL volumetric concentration is 25-35%2O2Solution continues to stir Mix reaction 15-20min；
6) HCl solution that 60-100mL volumetric concentration is 8-12% is added, stands 4-5 days；
7) supernatant of solution is first removed, Zai Qu lower layer viscous fluid centrifugal treating freezes viscous fluid until viscous fluid is in neutrality It is dry, obtain graphene oxide sheet；
8) graphene oxide sheet is ground into graphene oxide powder, i.e. GO powder；
9) claim HA 150-200mg in container, dissolved with 10-15mL formamide, to container closure, 45-55 DEG C of oil bath is to molten Solution；
10) HA solution is cooled to room temperature, weighs EDC 500-540mg, NHS 300-320mg respectively, use 8-12mL, 3- respectively It is added in HA solution after the formamide vortex dissolution of 7mL, activation-COOH 25-35min is stirred at room temperature, the HA activated is molten Liquid；
11) it will be placed in container dissolved with the 10-15mL formamide solution of 1-1.5mL ethylenediamine, use constant pressure addition under condition of ice bath Funnel instills the HA solution of activation in gained ethylenediamine solution, and 50-70min is added dropwise under condition of ice bath；
12) reaction system is warmed to room temperature reaction 2-4h；
13) acetone that the quality of pre-cooling is 3-5 times of reaction solution is added under ice bath after reaction, cooling, crystallization is stood, precipitating, 0.22 μm of organic film filters, and acetone washing precipitating is removed precipitating, redissolved with water, dialysed in water, and every 6-8h replaces a water, It is freeze-dried after dialysis 48-60h up to intermediate product HA-NH2；
14) it weighs GO powder 45-60mg to be placed in container, 30-40mL formamide is added, ice bath is transferred to separately after visiting super 25-35min In one container, and previous container is rinsed with 20-30mL formamide and flushing liquor is transferred in latter container；
15) EDC 170-175mg, NHS 100-105mg are weighed, and is dissolved respectively with 5-10mL formamide, is then added into In the latter container of step 14), activation 10-20min, the GO solution activated is stirred at room temperature；
16) 90-135mg HA-NH is weighed2It is dissolved in 10-15mL formamide, and 180-270 μ L triethylamine, end to be activated is added It is added dropwise in the GO solution of activation afterwards, reacts at room temperature 20-30h；
17) quality that pre-cooling is added under ice bath after reaction is 3-5 times of acetone of reaction solution, cooling, and crystallization is stood, and is sunk It forms sediment, 0.22 μm of organic film filters, and acetone washing precipitating is removed precipitating, redissolved with water, dialysed in water, and every 4-6h replacement is primary Water, dialysis 48-60h obtain HA-GO solution；
18) adriamycin that 0.8-1mL concentration is 0.8-1.2mg/mL is taken to instill in HA-GO solution；It is protected from light stirring 24-30h, from The heart collects particle, saves after the material freeze of lower layer is dry, target product is made.
2. preparation method as described in claim 1, which is characterized in that in step 1), be ultrasonically treated 20-30min.
3. preparation method as described in claim 1, which is characterized in that in step 2), stirring rate 400-800rpm, stirring 20-40min。
4. preparation method as described in claim 1, which is characterized in that in step 6), during standing, every 1-2d removes supernatant Aliquots of deionized water is added in liquid.
5. preparation method as described in claim 1, which is characterized in that in step 7), be freeze-dried 24-48h.
6. preparation method as claimed in claim 1 or 5, which is characterized in that in step 7), centrifugal process specifically: exist respectively 5- is respectively centrifuged under 3500-4500rpm, 5500-6500rpm, 7500-8500rpm, 9000-11000rpm, 11000-13000rpm 15min removes supernatant every time and aliquots of deionized water is added after centrifugation.
7. preparation method as described in claim 1, which is characterized in that the use of specification is MWCO in step 13) and step 17) The bag filter of=10000-14000Da.
CN201910929625.9A 2019-09-27 2019-09-27 It is a kind of using hyaluronic acid decorated graphene oxide as the preparation method of the Adriamycin nanoparticles of carrier Pending CN110507635A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910929625.9A CN110507635A (en) 2019-09-27 2019-09-27 It is a kind of using hyaluronic acid decorated graphene oxide as the preparation method of the Adriamycin nanoparticles of carrier

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910929625.9A CN110507635A (en) 2019-09-27 2019-09-27 It is a kind of using hyaluronic acid decorated graphene oxide as the preparation method of the Adriamycin nanoparticles of carrier

Publications (1)

Publication Number Publication Date
CN110507635A true CN110507635A (en) 2019-11-29

Family

ID=68633980

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910929625.9A Pending CN110507635A (en) 2019-09-27 2019-09-27 It is a kind of using hyaluronic acid decorated graphene oxide as the preparation method of the Adriamycin nanoparticles of carrier

Country Status (1)

Country Link
CN (1) CN110507635A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111298117A (en) * 2020-04-18 2020-06-19 中山市君泽科技有限公司 Method for treating cancer by combining graphene

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102727901A (en) * 2012-07-12 2012-10-17 上海师范大学 Graphene oxide/hyaluronic acid nanometer drug carrier material, preparation method and application of graphene oxide/hyaluronic acid nanometer drug carrier material
CN103030140A (en) * 2012-12-21 2013-04-10 郑州大学 Oxidized graphene modified by hyaluronic acid and preparation method and application of medicinal composition of oxidized graphene modified by hyaluronic acid
CN103980505A (en) * 2014-05-27 2014-08-13 武汉大学 Method for preparing hyaluronic acid/ graphene oxide nano composite hydrogel and medicinal composition
CN104189917A (en) * 2014-08-13 2014-12-10 东华大学 Preparation method of doxorubicin-containing graphene oxide medicine-carrying composite material
WO2017031060A1 (en) * 2015-08-14 2017-02-23 Board Of Regents Of The University Of Nebraska Substrate delivery of embedded liposomes
CN106809828A (en) * 2017-01-18 2017-06-09 上海应用技术大学 A kind of graphene oxide particle for target administration and preparation method thereof
CN109125272A (en) * 2018-08-23 2019-01-04 浙江理工大学 It is a kind of with fluorinated graphene be the carrier loaded nanoparticle for having mitoxantrone preparation method

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102727901A (en) * 2012-07-12 2012-10-17 上海师范大学 Graphene oxide/hyaluronic acid nanometer drug carrier material, preparation method and application of graphene oxide/hyaluronic acid nanometer drug carrier material
CN103030140A (en) * 2012-12-21 2013-04-10 郑州大学 Oxidized graphene modified by hyaluronic acid and preparation method and application of medicinal composition of oxidized graphene modified by hyaluronic acid
CN103980505A (en) * 2014-05-27 2014-08-13 武汉大学 Method for preparing hyaluronic acid/ graphene oxide nano composite hydrogel and medicinal composition
CN104189917A (en) * 2014-08-13 2014-12-10 东华大学 Preparation method of doxorubicin-containing graphene oxide medicine-carrying composite material
WO2017031060A1 (en) * 2015-08-14 2017-02-23 Board Of Regents Of The University Of Nebraska Substrate delivery of embedded liposomes
CN106809828A (en) * 2017-01-18 2017-06-09 上海应用技术大学 A kind of graphene oxide particle for target administration and preparation method thereof
CN109125272A (en) * 2018-08-23 2019-01-04 浙江理工大学 It is a kind of with fluorinated graphene be the carrier loaded nanoparticle for having mitoxantrone preparation method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
陈芊等: ""Hummers法制备氧化石墨烯"", 《四川化工》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111298117A (en) * 2020-04-18 2020-06-19 中山市君泽科技有限公司 Method for treating cancer by combining graphene

Similar Documents

Publication Publication Date Title
Liu et al. 2D magnetic titanium carbide MXene for cancer theranostics
Tian et al. Facile fabrication of rare-earth-doped Gd2O3 hollow spheres with upconversion luminescence, magnetic resonance, and drug delivery properties
Tian et al. Poly (acrylic acid) bridged gadolinium metal–organic framework–gold nanoparticle composites as contrast agents for computed tomography and magnetic resonance bimodal imaging
Sitharaman et al. Gd@ C60 [C (COOH) 2] 10 and Gd@ C60 (OH) x: Nanoscale aggregation studies of two metallofullerene MRI contrast agents in aqueous solution
Zhou et al. Dual-modality in vivo imaging using rare-earth nanocrystals with near-infrared to near-infrared (NIR-to-NIR) upconversion luminescence and magnetic resonance properties
Liu et al. Hybrid mesoporous gadolinium oxide nanorods: a platform for multimodal imaging and enhanced insoluble anticancer drug delivery with low systemic toxicity
CN102569749B (en) Graphene/hydroxyapatite nano composite and preparation method thereof
Daryan et al. Surface engineered hollow hydroxyapatite microspheres: Hydrothermal synthesis and growth mechanisms
Tian et al. Template-directed synthesis, properties, and dual-modal bioapplications of multifunctional GdPO4 hierarchical hollow spheres
CN110507635A (en) It is a kind of using hyaluronic acid decorated graphene oxide as the preparation method of the Adriamycin nanoparticles of carrier
CN102786090B (en) Method for synthesizing vanadic acid zinc overlength nanowire material by microwave radiation
Chen et al. Multifunctional PVP-Ba2GdF7: Yb3+, Ho3+ coated on Ag nanospheres for bioimaging and tumor photothermal therapy
Lv et al. Facile preparation of controllable-aspect-ratio hydroxyapatite nanorods with high-gravity technology for bone tissue engineering
Wang et al. Hydrothermal synthesis and structure characterization of flower-like self assembly of silicon-doped hydroxyapatite
Qian et al. Synthesis of urchin-like nickel nanoparticles with enhanced rotating magnetic field-induced cell necrosis and tumor inhibition
Huang et al. In Vivo MRI and X‐Ray Bifunctional Imaging of Polymeric Composite Supplemented with GdPO4· H2O Nanobundles for Tracing Bone Implant and Bone Regeneration
Zhao et al. Bottom-up synthesis of ultra-small molybdenum disulfide-polyvinylpyrrolidone nanosheets for imaging-guided tumor regression
CN107929242B (en) Drug carrier based on nano-diamond, drug complex, preparation method and application thereof
Scialla et al. Simplified preparation and characterization of magnetic hydroxyapatite-based nanocomposites
CN102552945B (en) Surface modification method of magnetic iron oxide nano-particles
Hamdan et al. Strategies for controlled synthesis of nanoparticles derived from a group of uniform materials based on organic salts
CN111228489A (en) Ferroferric oxide bismuth sulfide nano composite material and preparation and application thereof
CN106963951B (en) Graphene oxide/manganese tungstate/polyethylene glycol nano hybrid material and preparation thereof
Wu et al. Fabrication of Gd/Eu-codoped SmPO4 nanorods for dual-modal magnetic resonance and bio-optical imaging
Li et al. Effect of surface functionalities on relaxometric properties of MR contrast agents based on NaGdF 4 nanoparticles

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20191129